# Introduction

## Guide to RNA-seq Data Analysis

**I-Hsuan Lin**

The University of Manchester

<div align="left"><img src="https://808500276-files.gitbook.io/~/files/v0/b/gitbook-legacy-files/o/assets%2F-M5W-32iOTUXPKbX19Gv%2F-M7DmQoobN-srIkv54Uc%2F-M7Duft3VcAXdlFHMDHz%2FTAB_col_white_background.png?alt=media&#x26;token=65d73b02-2c69-43d9-8128-58abe91dcd0e" alt=""></div>

RNA-seq utilizes the sequencing technology to assay the presence and quantity of RNA molecules in the given sample. RNA-seq offers many advantages and supersede the microarray technology that was introduced in 2000s. This includes detects known and novel transcripts, increased specificity and sensitivity, and identification of low-abundance transcripts and isoforms with sufficient sequencing depth. RNA-seq has also been used to discover alternative splicing variants, chimeric RNAs result from fusion genes and RNA editing sites. We can compare RNA-seq data between conditions to detect differences across groups of samples in terms of (1) gene-level expression, (2) transcript/isoform-level expression, and (3) transcript/isoform usage within a gene.

I will use a real-world illumina paired-end RNA sequencing dataset to demonstrate a step-by-step guide where readers can reproduced the analysis. The complete workflow includes:&#x20;

* Performing QC and trimming to pre-process RNA-seq raw data
* Mapping of trimmed reads using either alignment-free (Salmon) and alignment-based (STAR) methods
* Quantifing gene and transcript expression
* Performing statistical testing to identified differentially expressed genes, transcripts and also detected expression switching between transcripts

## License

Unless specified, this content is licensed under the Creative Commons — Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). You may view a copy of this license at [https://creativecommons.org/licenses/by-nc-sa/4.0/](https://creativecommons.org/licenses/by-nc-sa/4.0/)

![](https://808500276-files.gitbook.io/~/files/v0/b/gitbook-legacy-files/o/assets%2F-M5W-32iOTUXPKbX19Gv%2F-M7CjNmaLtD8XJ1bcdBD%2F-M7DVraGBLyVDhgZ1eTe%2F200px-CC-BY-NC-SA.svg.png?alt=media\&token=f9d7d2c1-bfad-4f29-8e47-20e18c83c64e)
